The clinical hallmark of the genetic disorder cystic fibrosis (CF) is chronic pulmonary infection, particularly with Pseudomonas aeruginosa. The global objective of the proposed project is to gain further insight into the link between defects in the CF gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), and the pathogenesis of pulmonary infections with P. aeruginosa in CF patients. Preliminary studies in a CF cell culture model using the TUNEL assay indicates that P. aeruginosa induces apoptosis in normal, but not CF, lung cells. The specific objective of this project is to delineate the role of apoptosis in the interaction of P. aeruginosa with respiratory epithelium. The specific aims are twofold: 1) characterize the induction of apoptosis in cell culture and mouse models of CF following infection with P. aeruginosa, using TUNEL staining, FACS analysis and confocal microscopy of normal and CF lung cells, a. specifically, to detail the time-course of infection induced apoptosis, b. establish the role of internalization of P. aeruginosa in the induction of apoptosis, and c. determine the role of caspases in the infection-induced apoptosis, and 2) compare the expression of genes regulating apoptosis in lung cells expressing differing Cftr alleles, both before and after infection with P. aeruginosa, using DNA-microarray technology, a. specifically, to identify the expression levels of genes instrumental in orchestrating apoptosis in both wild-type and deltaF508 CFTR expressing lung cells after infection with P. aeruginosa, and b. determine the time course of expression of infection induced genes. This approach will allow simultaneous monitoring of the expression levels of hundreds of genes, potentially identifying and implicating cellular pathways previously unappreciated as participants in the process of respiratory cell-pseudomonal interaction. Thus, we propose to investigate the role of apoptosis in the pathogenesis of pseudomonal pulmonary infection in CF with the ultimate goal of perhaps formulating new treatment strategies based on the findings of these studies. Completion of this project promises to provide the experience necessary for the applicant to initiate a career as an independent investigator in the field of pseudomonal pathogensis, and to provide further insight in the pathogenesis of CF lung diseases.